In an effort to better understand the relationship between influenza virus tropism and the development of antiviral immunity we developed novel platform to restrict virus replication in a cell-specific manner. This involved insertion of endogenous, cell-specific microRNAs target sites into influenza virus mRNA, resulting in attenuation only in the presence of the miRNA. Using a virus restricted from replication in all cells of the immune system allowed for the discovery that the innate cytokine response relies on infection of immune cells. However the precise immune cells required for this remain unknown. This proposal will test the hypothesis that direct infection of dendritic cells and macrophages wil be required for the orchestration of antiviral cytokine responses. To address this hypothesis Aim 1 will generate a library of influenza viruses targeted by cell-specific microRNAs including those restricted to: myeloid cells (dendritic cells and macrophages), lymphoid cells (B and T cells), endothelial cells, and ciliated epithelial cells. As a control, viruses will be designed to be targeted in all mammalian cells, and an untargeted scrambled virus as a negative control. These viruses will be used to define the infected cell types required for orchestrating antiviral cytokin responses. In addition to cells directly infected with influenza, non-infected cells also participae in the immune response through bystander activation. To better define the role of direct infection Aim 2 will generate a panel of influenza viruses that express siRNAs designed to target host cytokines. Therefore, specific cytokines will be blunted only in cells directly infected with influenza virus. These recombinant viruses will be used to define the direct infection requirements for the induction of specific cytokines in vivo. The aims of this proposal use complementary approaches, through the exploitation of microRNAs, to define the role of virus tropism on the induction of antiviral immunity. These studies will elucidate key insights into the immune response and pathogenesis of influenza viruses and provide the basis for future rational vaccine and antiviral design.